Registration Dossier

Administrative data

Description of key information

The results of an in vitro skin irritation test in the EPISKIN model with Reactive Red F03-0318 indicated that the test item is non irritant. According to the results of an acute eye irritation study performed in New Zealand White rabbits, Reactive Red F03-0318 should not be classified as an eye irritant.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 March 2011 to 01 April 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: study conducted to EU & OECD test guidance in compliance with GLP and reported with a valid GLP certificate.
Qualifier:
according to
Guideline:
other: Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), ANNEX III, B.46. IN VITRO SKIN IRRITATION: RECONSTRUCTED HUMAN EPIDERMIS MODEL TEST
Qualifier:
according to
Guideline:
other: OECD Guidelines for Testing of Chemicals, Section 4, No. 439, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” adopted 22 July 2010.
GLP compliance:
yes (incl. certificate)
Species:
human
Strain:
not specified
Details on test animals and environmental conditions:
Human Skin
EpiSkinTM Small Model (EpiSkinTMSM), EPISKIN SNC Lyon, France, is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1984). Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.
Source: Skinethic, Nice, France.
Batch No.: 11-EKIN-013
Expiry date: 04 April 2011
Type of coverage:
open
Preparation of test site:
other: Not applicable
Vehicle:
water
Controls:
yes
Amount / concentration applied:
First, 10 µl distilled water (Supplier: HungaroPharma; Batch No.: 803 0211; Exp. date: 22 August 2011) was applied to the epidermal surface (in order to improve further contact between powder and epidermis) and then 10 mg of the test item was applied evenly to the epidermal surface.
Duration of treatment / exposure:
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes
Observation period:
42 hours
Number of animals:
Not applicable
Details on study design:
Preparation of the Test Item
The test item was applied in its original form, no formulation was required.

Controls
Positive and negative controls, furthermore additional controls for coloured substance were included in the experiment.

Negative Control
Phosphate Buffered Saline (PBS):
Supplier: SIGMA-ALDRICH
Batch No.: BCBC6808
Expiry date: December 2014

Positive Control
SDS 5 % aq. solution (prepared in LAB Research Ltd.):
Sodium Dodecyl Sulphate (SDS)
Supplier: SIGMA-ALDRICH
Batch No.: 129K0187
Expiry date: December 2012 Distilled water
Supplier: Hungaropharma Zrt
Batch No.: 803 0211
Expiry date: 22 August 2011

Quality Control
EPISKIN-SM kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking an MTT cell viability test and a cytotoxicity test with sodium dodecyl sulphate (SDS).

Justification for Selection of the Test System
The EPISKIN model has been validated for irritation testing in an international trial, it is considered to be suitable for this study.

DEMONSTRATION OF PROFICIENCY
Prior to routine use of the method LAB Research Ltd. demonstrated technical proficiency, using the ten Proficiency Chemicals according to OECD Test Guideline No. 439.

Kit Content
Units: EPISKIN-SM plate containing up to 12 reconstructed epidermis units (area: 0.38 cm2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.
Plate: 12-well assay plate
Punch: EPISKIN-SM biopsy punch for easy sampling of epidermis
Medium: A flask of sterile “Maintenance Medium” for incubations. (Batch No.: 11-MAIN3-013; Exp. Date: 06 April 2011)
A flask of sterile “Assay Medium” for use in for use in MTT assays.(Batch No.: 11-ESSC-013; Exp. Date: 06 April 2011)

Number of Replicate Wells
In this assay 3 replicates for the test item and 3 negative controls + 3 positive controls were used. Furthermore 2 replicates of additional controls for coloured substance were used.

Kit Reception Quality Check
The colour of the agar medium used for transport was checked for its pH:
- orange colour = good
- yellow or violet colour = not acceptable

The colour of the temperature indicator was inspected to verify that the kit has not been exposed to a temperature above 40°C:
- the indicator changes from white to grey at 40°C

The kit was found to be in good order at reception.

Storage
The EPISKIN-SM kit was kept in its packaging at room temperature and the assay and maintenance medium supplied with the kit was stored at 2-8°C until the initiation of the test.

Additional materials

MTT stock solution
MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1] was dissolved to a final concentration of 3 mg/mL in saline buffer (PBS). The obtained stock solution can be stored in a refrigerator (2-8°C), protected from light up to 15 days.

MTT ready to use solution
The MTT stock solution was diluted with pre-warmed “assay medium” to a final concentration of 0.3 mg/mL. The obtained solution was used within one hour.

Acidified isopropanol
Isopropanol was diluted with HCl acid to a final concentration of 0.04N HCl.
The obtained solution can be stored in a refrigerator (2-8°C), protected from light for one month.

INDICATOR FOR POTENTIAL FALSE VIABILITY
Optical properties of the test material or its chemical action on MTT may interfere with the assay leading to a false estimate of viability. This may occur when the test substance is not completely removed from the tissue by rinsing or when it penetrates the epidermis. If the test material acts directly on MTT (MTT-reducer), is naturally coloured, or becomes coloured during tissue treatment, additional controls will be used to detect and correct for test substance interference with the viability measurement. Methods of how to correct direct MTT reduction and interferences by colouring agents are detailed below.

Check-method for possible direct MTT reduction with test substance
An amount of 10 mg test item was added to 2 mL MTT ready to use solution and mixed. The mixture was incubated for three hours at room temperature protected from light and then any colour change was assessed:

- Test substances which do not interact with MTT: yellow
- Test substances interacting with MTT: blue or purple

If the MTT solution colour becomes blue or purple, the test substance interacts with the MTT. It is then necessary to evaluate the part of optical density (OD) due to the non specific reduction of the MTT (i.e. by using killed epidermis).

The test item showed no direct interaction with MTT.
Check-method to detect the colouring potential of test-substances

Prior to treatment, chemicals were evaluated for their intrinsic colour or ability to become coloured in contact with water (simulating a tissue humid environment).
As the test item has an intrinsic colour, further evaluation to detect colouring potential was not necessary. Non Specific Colour % (NSC %) was determined in order to evaluate the ability of test substance to stain the epidermis by using additional control tissues.

Additional control(s) for dyes and chemicals able to colour the tissue
In addition to the normal procedure, three additional chemical-treated tissues were used for the non specific OD evaluation. These tissues followed the same treatment steps as the other tissues except for the MTT step: MTT incubation was replaced by incubation with fresh assay medium. OD readings were made following the same conditions as for the other tissues.

PERFORMANCE OF THE STUDY
Procedures were performed under axenic conditions.

Pre-incubation (day [-1])
The “maintenance medium” was pre-warmed to 37°C. The appropriate number of assay plate wells were filled with the pre-warmed medium (2 mL per well). The epidermis units were placed, with the media below them in contact with the epidermis, into each prepared well and then incubated overnight at 37°C in an incubator with 5% CO2.

Application and rinsing (day 0)
Test Item
First, 10 µl distilled water (Supplier: HungaroPharma; Batch No.: 803 0211; Exp. date: 22 August 2011) was applied to the epidermal surface (in order to improve further contact between powder and epidermis) and then 10 mg of the test item was applied evenly to the epidermal surface. Test substance was spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary.

Positive and negative control
An amount of 10 µl positive (SDS) or negative (PBS) control were added to each of three test skin units by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary.

Additional controls for staining effects
These tissues followed the same application procedure as the other Test Item treated tissues.
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature (18-28°C).

After the incubation time the EPISKIN-SM units were removed and rinsed thoroughly with PBS 1x solution (0.9%) to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with a suitable pipette tip linked to a vacuum source (care was taken to avoid the damage of epidermis).

After rinsing the units were placed into the plate wells with fresh pre-warmed “maintenance medium” (2 mL/well) below them and then incubated for 42 hours (± 1h) at 37°C in an incubator with 5% CO2.

MTT test after 42 hours incubation (day 2)
After the 42 hours incubation all EPISKIN-SM units except the 3 staining controls were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well). The three additional controls for coloured substances were transferred to wells filled with fresh assay medium. Then, all transferred EPISKIN-SM units were incubated for 3 hours (± 5 min) at 37°C in an incubator with 5% CO2 protected from light.

Formazan extraction (day 2
At the end of incubation with MTT a formazan extraction was undertaken:
A disk of epidermis from each replicate was cut from the unit (this involves the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube of 500 µL acidified isopropanol (one tube corresponding to one well of the tissue culture plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material with the acidified isopropanol then incubated for about two hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.

Cell viability measurements (day 2)
Following the formazan extraction, 2×200 µL samples from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD (Absorbance / Optical Density) of the samples in a 96-well plate spectrophotometer was read at 540 nm using acidified isopropanol solution blank (6×200 µL).

Note: The validity of the microplate reader was verified with a standard verification plate daily before use. The standard plate was calibrated yearly by the manufacturer.
Irritant / corrosive response data:
VALIDITY OF THE TEST
The mean OD value of the three negative control tissues was 0.639. The positive control result showed 9 % viability. Each standard deviation value (SD) of the % viability was below 18. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

INDICATOR FOR POTENTIAL FALSE VIABILITY

Possible direct MTT reduction with test substance
No colour change was observed after three hours of incubation. The test material did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

Colouring potential of test substances
As the test item has an intrinsic colour, three additional chemical-treated tissues were used for the non specific OD evaluation. Mean OD (measured at 540 nm) of these tissues were determined as 0.018, Non Specific Colour % was calculated as 2.76%. Therefore additional data calculation was not necessary. A false estimation of viability can be precluded.

Cell VIABILITY

 

The results of the optical density (OD) measured at 540 nm of each replicate and the calculated % viability of the cells is presented below:

 

Substance

Optical Density (OD)

Viability (%)

Negative Control:

PBS

 

1

0.668

104

2

0.642

100

3

0.608

95

mean

0.639

100

standard deviation (SD)

4.71

Positive Control:

SDS 5% aq. solution

 

1

0.080

13

2

0.041

6

3

0.051

8

mean

0.058

9

standard deviation (SD)

3.18

Test Item:

Reactive Red F03-0318

 

1

0.565

88

2

0.534

83

3

0.484

76

mean

0.528

83

standard deviation (SD)

6.39

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In this in vitro skin irritation test in the EPISKIN model with Reactive Red F03-0318 the results indicated that the test item is Non Irritant (NI) [UN GHS: No Category].
Executive summary:

This study has been performed in accordance with the study plan the OECD Guidelines for Testing of Chemicals (No. 439, 22 July 2010), EpiSkin™ SOP, Version 1.8 (February 2009), COMMISSION REGULATION (EC) No 761/2009 ANNEX III, B.46 and the Principles of Good Laboratory Practice (GLP) and reported with a valid GLP certificate.

 

Disks of EPISKIN (three units / chemical) were treated with Reactive Red F03-0318 and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution (0.9%). Epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution atin 5% CO2 protected from light. The formazan extract in acidified isopropanol was then spectrophotometrically evaluated for optical density (OD) and quantified.

SDS 5% and PBS treated epidermis were used as positive and negative controls, respectively. For each treated tissue viability was expressed as a % relative to negative control.

The test substance is considered to be irritant to skin, if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.

 

In this in vitro skin irritation test in the EPISKIN model with Reactive Red F03-0318 the results indicated that the test item is Non Irritant (NI) [UN GHS: No Category].

 

All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 March 2011 to 12 April 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: study conducted to EU & OECD test guidance in compliance with GLP and reported with a valid GLP certificate.
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
yes
Remarks:
The relative humidity (min 24%) was out of the target range (30-70%) during the study.
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
yes
Remarks:
The relative humidity (min 24%) was out of the target range (30-70%) during the study.
Principles of method if other than guideline:
The deviations are considered to have no impact on the outcome of the study and interpretation of the results.
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
Species and strain: New Zealand White rabbits
Source: S&K-LAP Kft. 2173 Kartal, Császár út 135, Hungary
Justification of strain: The New Zealand White rabbit is one of the standard strains used for acute irritation toxicity studies.
Animal health: Only animals in acceptable health condition were used for the test. Both eyes of each animal provisionally selected for testing were examined approximately one hour before starting the study. Animals showing eye irritation, ocular defects or pre-existing corneal injury were not used.
Number of animals: 3 animals
Age of animals at treatment: ~12 weeks old (adult)
Sex: Male
Body weight range at the
beginning of the life phase: 2923 – 3311 g
end of the life phase: 3514 – 3996 g
Date of receipt: 09 March 2011
Acclimatization time: 13 days
Animal identification: The individual identification was by engraved ear tag. The cages were marked with individual identity cards with information about study code, sex, dose group, cage number and individual animal number.


HUSBANDRY

Animal health: Only healthy animals were used for the test. The veterinarian certified health status.
Number of animal room: 620
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 20 ± 3°C
Relative humidity: 24 - 63 %
Housing/Enrichment: Rabbits were individually housed in AAALAC approved metal wire rabbit cages. Cages were of an open wire structure and cages were placed together to allow some social interaction with rabbit(s) in adjoining cages
Ventilation: 15-20 air exchanges/hour

The environmental parameters were recorded twice daily during the study.

FOOD AND FEEDING

Animal received PURINA Base – Lap gr. diet for rabbits produced by AGRIBRANDS Europe Hungary PLC, H-5300 Karcag, Madarasi road, Hungary, ad libitum.

WATER SUPPLY AND QUALITY CONTROL OF WATER

The animals received municipal tap water, as for human consumption, ad libitum, from an automatic system.

The quality control analysis is performed once every three months and microbiological assessment is performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36., Hungary). The quality control results are retained in the archives of LAB Research Ltd.
Vehicle:
unchanged (no vehicle)
Controls:
other: untreated right eye served as control
Amount / concentration applied:
A single dose of 0.1 g of the solid test item Reactive Red F03-0318 was administered to each animal.
Duration of treatment / exposure:
The eyes of the test animals were washed out at 1 hour after application of test item using physiological saline solution.
Observation period (in vivo):
72 hours
Number of animals or in vitro replicates:
3 male
Details on study design:
Three male animals in acceptable health condition were selected for the test. Care was taken to select only those animals that had a normal eye condition and any with ocular lesions were rejected.

An initial test was performed using one animal. The test item was instilled into the conjunctival sac of the left eye. The eyelids were held closed for several seconds to prevent the loss of the test item. The contralateral eye served as the control. Immediately after the administration of the test item, an assessment of the initial pain reaction was made according to a six point scale.

After consideration of the ocular responses produced in the first animal, two additional animals were treated.

OBSERVATIONS AND SCORING

Clinical Observations and Eye Examination

The eyes were examined at 1, 24, 48, 72 hours and 1, 2 and 3 weeks after treatment. The duration of the observation period was sufficient to identify reversibility or irreversibility of changes. Any clinical signs of toxicity or signs of ill- health during the study were recorded.

At the end of the observation period, each animal was sacrificed by intramuscular injections of CP-Ketamin 10% and CP-Xylazin 2% followed by iv. Euthasol® 40% anaesthesia. Death was verified by checking pupil and corneal reflex and the absence of respiration.

Scoring and Assessment of Local Reaction

The eye irritation scores were evaluated according to the scoring system by Draize (1977) and OECD 405 (24 April 2002).

Classification of the Test Items

Individual reactions of the animal were recorded at each observation time. The nature, severity and duration of all lesions observed were described.

Results were presented and interpreted according to Regulation (EC) No 1272/2008, as follows:

Irreversible effects on the eye/serious damage to eyes (Category 1)

Substances that have the potential to seriously damage the eyes are classified in Category 1 (irreversible effects on the eye). These observations include animals with grade 4 cornea lesions and other severe reactions (e.g., destruction of cornea) observed at any time during the test, as well as persistent corneal opacity, discoloration of the cornea by a dye substance, adhesion, pannus, and interference with the function of the iris or other effects that impair sight.

Category for irreversible eye effects
If, when applied to the eye of an animal, a substance produces:
— at least in one animal effects on the cornea, iris or conjunctiva that are not expected to reverse or have not fully reversed within an observation period of normally 21 days;
and/or
— at least in 2 of 3 tested animals, a positive response of:
o corneal opacity ≥ 3 and/or
o iritis > 1.5
calculated as the mean scores following grading at 24, 48 and 72 hours after installation of the test material.

Reversible effects on the eye/irritating to eyes (Category 2)

Substances that have the potential to induce reversible eye irritation are classified in Category 2 (irritating to eyes).

Category for reversible eye effects
If, when applied to the eye of an animal, a substance produces:
— at least in 2 of 3 tested animals, a positive response of:
o corneal opacity ≥ 1 and/or
o iritis ≥ 1, and/or
o conjunctival redness ≥ 2 and/or
o conjunctival oedema (chemosis) ≥ 2

— calculated as the mean scores following grading at 24, 48 and 72 hours after installation of the test material, and which fully reverses within an observation period of 21 days

Measurement of Body Weight

Individual body weight was recorded at the beginning and end of the study.


Irritation parameter:
cornea opacity score
Basis:
animal #1
Remarks:
mean
Time point:
other: Overall at 24, 48 and 72 hours
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
cornea opacity score
Basis:
animal #2
Remarks:
mean
Time point:
other: Overall at 24, 48 and 72 hours
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
cornea opacity score
Basis:
animal #3
Remarks:
mean
Time point:
other: Overall at 24, 48 and 72 hours
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
iris score
Basis:
animal #1
Remarks:
mean
Time point:
other: Overall at 24, 48 and 72 hours
Score:
0
Max. score:
2
Reversibility:
other: not applicable
Irritation parameter:
iris score
Basis:
animal #2
Remarks:
mean
Time point:
other: Overall at 24, 48 and 72 hours
Score:
0
Max. score:
2
Reversibility:
other: not applicable
Irritation parameter:
iris score
Basis:
animal #3
Remarks:
mean
Time point:
other: Overall at 24, 48 and 72 hours
Score:
0
Max. score:
2
Reversibility:
other: not applicable
Irritation parameter:
conjunctivae score
Basis:
animal #1
Remarks:
mean
Time point:
other: Overall at 24, 48 and 72 hours
Score:
2
Max. score:
3
Reversibility:
fully reversible within: 3 weeks
Irritation parameter:
conjunctivae score
Basis:
animal #2
Remarks:
mean
Time point:
other: Overall at 24, 48 and 72 hours
Score:
1
Max. score:
3
Reversibility:
fully reversible within: 3 weeks
Irritation parameter:
conjunctivae score
Basis:
animal #3
Remarks:
mean
Time point:
other: Overall at 24, 48 and 72 hours
Score:
1.33
Max. score:
3
Reversibility:
fully reversible within: 3 weeks
Irritation parameter:
chemosis score
Basis:
animal #1
Remarks:
mean
Time point:
other: Overall at 24, 48 amd 72 hours
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
chemosis score
Basis:
animal #2
Remarks:
mean
Time point:
other: Overall at 24, 48 and 72 hours
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
chemosis score
Basis:
animal #3
Remarks:
mean
Time point:
other: Overall at 24, 48 and 72 hours
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 hours
Irritant / corrosive response data:
Initial Pain Reaction (IPR) (score 3) was observed in all animals.

One hour after the application:

Conjunctival redness (score 1) and different severities of conjunctival discharge (score 2 or 3) were found in all rabbits. Two animals showed conjunctival chemosis (score 2 or 3). The conjunctiva was coloured by the test item in all animals, the cornea in one rabbit. The cornea discoloration persisted up to and including the 72 hour observation, the conjunctiva discoloration was not longer present at the 3 weeks observation.

At 24 hours after treatment:

Conjunctival redness (score 2 or 1) was found in two and one rabbit, respectively, and discharge (score 1) was observed in all animals. Two rabbits displayed conjunctival chemosis (score 1).

At 48 and 72 hours and 1 week after treatment:

Conjunctival redness (score 22 or 1) was seen in one rabbit and two rabbits, respectively.

At 2 weeks after treatment, conjunctival redness (score 1) was found in all rabbits.

At 3 weeks after treatment neither signs of eye irritation nor discoloration of the tissues of the eyes were observed.

The study was terminated after the 3 weeks observation.

During the study, no signs of eye irritation were observed in the control eye of all animals.
Other effects:
There was no mortality observed during the study.
The body weights and body weight changes were considered to be normal with no indication of treatment related effect.
The general state and behavior of animals were normal throughout the study period. There were no clinical signs observed that could be related to treatment.

INDIVIDUAL SCORES FOR OCULAR IRRITATION

 

 

Study Code:             11/008-005N                    Species:    NZW Rabbit

Dose:                        0.1 g                                 Sex:          Male

Start of Exposure:    22 March 2011                  Test Item: Reactive Red F03-0318

 

 

Abbreviations:   R    = Redness                                OD =   Opacity degree of density

                            CH = Chemosis                              OE =   Extentof opaque area

D    = Discharge                             IPR=   Initial pain reaction

 

 

Time

Animal No.

Score of irritation

IPR

Conjunctivae

Opacity of cornea

Iris

Control eye

Other sign

R

CH

D

OD

OE

R

1 hour

00008

1

2

3

0

0

0

0

*

3

00009

1

0

2

0

0

0

0

*

3

00003

1

3

3

0

0

0

0

**

3

Time of Observation: Day 0

Time

Animal No.

Score of irritation

Conjunctivae

Opacity of cornea

Iris

Control eye

Other sign

R

CH

D

OD

OE

R

24 hours

00008

2

1

1

0

0

0

0

*

00009

1

0

1

0

0

0

0

*

00003

2

1

1

0

0

0

0

**

Time of Observation: Day 1

Time

Animal No.

Score of irritation

Conjunctivae

Opacity of cornea

Iris

Control eye

Other sign

R

CH

D

OD

OE

R

48 hours

00008

2

0

0

0

0

0

0

*

00009

1

0

0

0

0

0

0

*

00003

1

0

0

0

0

0

0

**

Time of Observation: Day 2

*: The conjunctiva was coloured by the test item.

**: The conjunctiva and the cornea were coloured by the test item.

 

Time

Animal No.

Score of irritation

Conjunctivae

Opacity of cornea

Iris

Control eye

Other sign

R

CH

D

OD

OE

R

72 hours

00008

2

0

0

0

0

0

0

*

00009

1

0

0

0

0

0

0

*

00003

1

0

0

0

0

0

0

**

 

Time of Observation: Day 3

 

Time

Animal No.

Score of irritation

Conjunctivae

Opacity of cornea

Iris

Control eye

Other sign

R

CH

D

OD

OE

R

1 week

00008

2

0

0

0

0

0

0

*

00009

1

0

0

0

0

0

0

*

00003

1

0

0

0

0

0

0

*

 

Time of Observation: 1 week

 

*: The conjunctiva was coloured by the test item.

**: The conjunctiva and the cornea were coloured by the test item.

Time

Animal No.

Score of irritation

Conjunctivae

Opacity of cornea

Iris

Control eye

Other sign

R

CH

D

OD

OE

R

2 weeks

00008

1

0

0

0

0

0

0

*

00009

1

0

0

0

0

0

0

*

00003

1

0

0

0

0

0

0

*

 Time of Observation: 2 weeks

*: The conjunctiva was coloured by the test item.

 

Time

Animal No.

Score of irritation

Conjunctivae

Opacity of cornea

Iris

Control eye

Other sign

R

CH

D

OD

OE

R

3 weeks

00008

0

0

0

0

0

0

0

-

00009

0

0

0

0

0

0

0

-

00003

0

0

0

0

0

0

0

-

 Time of Observation: 3 weeks

 

MEAN VALUES OF EYE IRRITATION (24, 48, 72 hour reading)

Animal Number

Sex

Cornea Opacity

Iris

Conjunctivae

Redness

Chemosis

Discharge

00008

male

0.00

0.00

2.00

0.33

0.33

00009

male

0.00

0.00

1.00

0.00

0.33

00003

male

0.00

0.00

1.33

0.33

0.33

 

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item Reactive Red F03-0318, applied to rabbit eye mucosa, caused significant conjunctival irritant effects at one hour after application. The effects were fully reversible within 3 weeks.

According to Regulation (EC) No 1272/2008, REACTIVE RED F03-0318 does not require classification as an eye irritant.
Executive summary:

An acute eye irritation study of the test item Reactive Red F03-0318was performed in New Zealand White rabbits. The irritation effects of the test item were evaluated according to the Draize method (OECD No.: 405, 2002).

The test item was placed into the conjunctival sac of the left eye of each animal. The untreated right eye served as control. An amount ofof the test item was administered as a single dose.

 

Individual body weight was recorded at the beginning and end of the study. Morbidity and clinical signs of toxicity were checked daily.The eyes were examined at 1, 24, 48, 72 hours and 1, 2 and 3 weeks after the application.

 

No adverse effects on body weight development were noted during the study period. The general state and behavior of animals were normal throughout the study period.

 

Eye examination:

 

During the study, no signs of eye irritation were observed in the control eye of all animals.

 

Initial Pain Reaction(IPR) (score 3) was observed in all animals immediately after test item administration.

 

One hour after the application:

 

Conjunctival redness (score 1) and different severities of conjunctival discharge (score 2 or 3) were found in all rabbits. Two animals showed conjunctival chemosis (score 2 or 3). The conjunctiva was coloured by the test item in all animals, the cornea in one rabbit. The cornea discoloration persisted up to and including the 72 hour observation, the conjunctiva discoloration was not longer present at the 3 weeks observation.

At 24 hours after treatment:

 

Conjunctival redness (score 2 or 1) was found in two and one rabbit, respectively, and discharge (score 1) was observed in all animals. Two rabbits displayed conjunctival chemosis (score 1).

At 48 and 72 hours and 1 week after treatment:

 

Conjunctival redness (score 2 or 1) was seen in one rabbit and two rabbits, respectively.

 

At 2 weeks after treatment, conjunctival redness (score 1) was found in all rabbits.

 

At 3 weeks after treatment, neither signs of eye irritation nor discoloration of the tissues of the eyes were observed.

 

The study was terminated after the 3 weeks observation.

The animal’s individual mean scores (considering readings at 24, 48 and 72 hours after the treatment) were as follows:

 

Animal Number

Cornea Opacity

Iris

Conjunctivae

Redness

Chemosis

Discharge

00008

0.00

0.00

2.00

0.33

0.33

00009

0.00

0.00

1.00

0.00

0.33

00003

0.00

0.00

1.33

0.33

0.33

The test item REACTIVE RED F03-0318, applied to rabbit eye mucosa, caused significant conjunctival irritant effects at one hour application. The effects were fully reversible within 3 weeks.

According to Regulation (EC) No 1272/2008, REACTIVE RED F03-0318 does not require classification as an eye irritant.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation / Corrosion

The results of an in vitro skin irritation test in the EPISKIN model with Reactive Red F03-0318 indicated that the test item is non irritant.

Eye irritation

The results of the in vitro eye irritation study in the isolated chicken eyes model with Reactive Red F03-0318 indicated that the test item was not irritating.

The irritant effects of Reactive Red F03-0318 were evaluated according to the Draize method (OECD No.: 405, 2002) in an

acute eye irritation study performed in New Zealand White rabbits.

The test item Reactive Red F03-0318 applied to rabbit eye mucosa, caused significant conjunctival irritant effects at one hour application. The effects were fully reversible within 3 weeks.

Respiratory irritation

Respiratory irritation was not assessed; however no effects on the animals were noted in any associated studies.

The following information is taken into account for any hazard / risk assessment:

Skin and eye irritation are discussed.

Value used for CSA:

-         Skin irritation / corrosion: Not irritating

-         Eye irritation: Not irritating


Justification for selection of skin irritation / corrosion endpoint:
Only 1 study available.

Justification for selection of eye irritation endpoint:
In vivo study.

Justification for classification or non-classification

The above studies have all been ranked reliability 1 according to the Klimisch et al system. This ranking was deemed appropriate because the studies were conducted to GLP an in compliance with agreed protocols. Sufficient dose ranges and numbers are detailed; hence it is appropriate for use based on reliability and animal welfare grounds.

The above results triggered no classification under the Dangerous Substance Directive (67/548/EEC) and the CLP Regulation (EC No 1272/2008).